Mutagenesis of highly conserved histidines in lecithincholesterol acyltransferase: identification of an essential histidine (His 377).
Lecithin-cholesterol acyltransferase (LCAT) is responsible for the formation of cholesterol esters in plasma and is implicated in the removal of excess cholesterol from peripheral tissues. It is generally accepted that the catalytic mechanism of LCAT is similar to that of serine proteases and lipases involving a Ser, a His, and an acidic amino acid residue. Ser181 in LCAT has been previously identified as a catalytic residue; however, the active site His and acidic residue have not yet been identified. In this study we have used a variety of approaches to identify the putative active site histidine. Alignments of LCAT sequences across various species indicate that the four histidines at positions 180, 263, 368, and 377 are conserved and could be involved in catalysis. Based on the observation that the members of the triad preserve the same orientation in the primary sequence of a large number of lipases, we eliminated His180 as a potential candidate. Mutational analysis along with functional assays show that, in contrast to the replacement of His263 and His368, the replacement of the His at position 377 with Gly, Ala, or Ser obliterates LCAT activity with interfacial and water-soluble substrates, thus indicating a role of His377 in catalysis.
Adimoolam
S
S
Department of Biochemistry, College of Medicine at Urbana-Champaign, University of Illinois 61801, USA.
Lee
Y P
YP
Jonas
A
A
D000818:Animals; D001665:Binding Sites / Q000235:genetics; D002087:Butyrates / Q000378:metabolism; D019556:COS Cells; D017124:Conserved Sequence / Q000235:genetics; D006639:Histidine / Q000235:genetics; D008075:Lipoproteins, HDL / Q000378:metabolism; D008077:Lipoproteins, LDL / Q000378:metabolism; D016297:Mutagenesis, Site-Directed / Q000235:genetics; D007862:Phosphatidylcholine-Sterol O-Acyltransferase / Q000235:genetics* / Q000378:metabolism; D016415:Sequence Alignment; D014162:Transfection / Q000235:genetics
D002087:Butyrates; D008075:Lipoproteins, HDL; D008077:Lipoproteins, LDL; C033592:4-nitrophenyl butyrate; D006639:Histidine; D007862:Phosphatidylcholine-Sterol O-Acyltransferase